Antibacterial effect of a new haemostatic agent on - Medicina Oral ...

J Clin Exp Dent. 2012;4(3):e151-5. <strong>Antibacterial</strong> <strong>effect</strong> <strong>of</strong> <strong>haemostatic</strong> <strong>agent</strong>s.
Journal section: Clinical and Experimental Dentistry
Publication Types: Research
<strong>Antibacterial</strong> <strong>effect</strong> <strong>of</strong> a <strong>new</strong> <strong>haemostatic</strong> <strong>agent</strong> on oral microorganisms
Çağdaş Çinar 1 , Mesut Enes Odabaş 1 , Gülçin Akca 2 , Berrin Işik 3
1 Assistant pr<strong>of</strong>essor. Department <strong>of</strong> pediatric dentistry, faculty <strong>of</strong> dentistry, University <strong>of</strong> Gazi, Ankara, Turkey.
2 MD, PhD. Department <strong>of</strong> basic medical sciences, microbiology laboratory, faculty <strong>of</strong> dentistry, University <strong>of</strong> Gazi, Ankara,
Turkey.
3 MD, Associate pr<strong>of</strong>essor. Anesthesiology and reanimation specialist, department <strong>of</strong> anesthesiology and reanimation, faculty <strong>of</strong>
medicine, University <strong>of</strong> Gazi, Ankara, Turkey.
Correspondence:
Gazi Üniversitesi Dişhekimliği Fakültesi
Pedodonti Anabilim Dali Bişkek Cad. 82.Sok
06510 Emek-Ankara TURKEY
E-mail: mesut@gazi.edu.tr
Received: 24/11/2011
Accepted: 27/03/2012
Abstract
Çinar Ç, Odabaş ME, Akca G, Işik B. <strong>Antibacterial</strong> <strong>effect</strong> <strong>of</strong> a <strong>new</strong> <strong>haemostatic</strong>
<strong>agent</strong> on oral microorganisms. J Clin Exp Dent. 2012;4(3):e151-5.
http://www.medicinaoral.com/odo/volumenes/v4i3/jcedv4i3p151.pdf
Article Number: 50750 http://www.medicinaoral.com/odo/indice.htm
© Medicina Oral S. L. C.I.F. B 96689336 - eISSN: 1989-5488
eMail: jced@jced.es
Indexed in:
Scopus
DOI® System
Objective: The purpose <strong>of</strong> this study was to determine the antibacterial <strong>effect</strong> <strong>of</strong> a <strong>new</strong>ly developed <strong>haemostatic</strong>
<strong>agent</strong> Ankaferd Blood Stopper® (ABS) and Ferric Sulphate (FS) on various oral microorganisms.
Study design: Bacterial strains were freshly incubated in their specific broth media. For each <strong>of</strong> the strains, 3 wells
per each <strong>agent</strong>, with a 5 mm diameter were made under aseptic conditions in the specific agar media. Then they
were filled with a test <strong>agent</strong>s or 0.2% chlorhexidine digluconate (CHX) (control group). After 24h and 48h incubation
periods, inhibition zones were measured.
Results: ABS showed antibacterial <strong>effect</strong> on all test microorganisms except Lactobacillus acidophilus and Lactobacillus
salivarius. Ferric sulphate and CHX have antibacterial <strong>effect</strong> on all microorganisms. When the test <strong>agent</strong>s
compared, the inhibition zones <strong>of</strong> the ABS were found smaller than the ferric sulphate and CHX.
Conclusions: Although ferric sulphate and ABS have antibacterial <strong>effect</strong>, ferric sulphate had better antibacterial
activity than ABS on oral microorganisms under in vitro condition. FS and ABS not only exhibit the <strong>haemostatic</strong>
activity but also antimicrobial activity.
Key word: Ankaferd Blood Stopper, Ferric Sulphate, Haemostatic <strong>agent</strong>, Haemostasia, Bleeding, Bactericide.
e151
doi:10.4317/jced.50750
http://dx.doi.org/10.4317/jced.50750

J Clin Exp Dent. 2012;4(3):e151-5. <strong>Antibacterial</strong> <strong>effect</strong> <strong>of</strong> <strong>haemostatic</strong> <strong>agent</strong>s.
Introduction
Haemorrhage control is an essential factor in dental procedure
for good visualization <strong>of</strong> the fields. Dentists perform
variety operative procedures such as pulpotomy,
tooth extraction, biopsies, placement <strong>of</strong> endosseous implants,
periradicular and periodontal surgery, which may
require haemorrage control (1, 2). The most widespread
technique is to control bleeding by applying mechanical
pressure to wound surface. The use <strong>of</strong> cotton balls or
gauze pads risk leaving fibers in the surgical site. These
fibers may elicit a foreign body reaction and delay
healing (3). Another technique to control bleeding is the
application <strong>of</strong> <strong>haemostatic</strong> <strong>agent</strong>s(4). Various <strong>agent</strong>s are
used for hemorrhage control in dentistry.
Ferric sulphate (Fe 2 [SO 4 ] 3 ) has been used as a coagulative
and <strong>haemostatic</strong> <strong>agent</strong> and it was first used in medicine
in 1957. On contact with blood, the agglutination
<strong>of</strong> blood proteins result from the reaction <strong>of</strong> blood with
ferric and sulphate ions. Thus the capillary orifices have
been obturate by bonded proteins (3).
Medical plants have been marketed as antibacterial
<strong>agent</strong>s and <strong>haemostatic</strong> <strong>agent</strong>s for many years (5, 6).
Ankaferd Blood Stopper (ABS) (Ankaferd Sağlik Ürünleri
A.Ş., Istanbul, Turkey) is a mixture <strong>of</strong> herbal medicine
and it is recently developed to facilitate the rapid
hemorrhage control in difficult clinical conditions (6).
ABS ingredients a standard mixture <strong>of</strong> following plants:
Thymus vulgaris (5.0g/100ml), Glycyrrhiza glabra
(7.0g/100ml), Vitis vinifera (8.0g/100ml), Alpinia <strong>of</strong>ficinarum
(7.0g/100ml) and Urtica dioica (6.0g/100ml).
ABS stimulated rapid formation <strong>of</strong> a protein network
and erythrocyte aggregation. The ABS-induced network
formation is related to the function <strong>of</strong> blood proteins and
red blood cells. The basic mechanism <strong>of</strong> action for ABS
appears to be the formation <strong>of</strong> an encapsulated protein
network that provides focal points for erythrocyte aggregation.
ABS also interacts with fibrinogen and other
blood proteins. Exposure to ABS seems to provide tissue
oxygenation as well as a physiological <strong>haemostatic</strong>
process without affecting any individual clotting factor.
This unique mechanism <strong>of</strong> action provides ABS with
an advantage (7). ABS has been safely used in patients
to treat epistaxis (8), after tonsillectomy (9) or variceal
bleeding(10). In addition, ABS has been used to control
upper gastrointestinal bleeding (11). Moreover the levels
<strong>of</strong> coagulation factors II, V, VIII, IX, X, XI, and XII
were not affected by ABS; therefore, ABS might be used
in patients with deficient primary haemostasis (12).
Many microorganism species have been detected in the
oral cavity; some <strong>of</strong> these bacterial species can cause
several diseases and complications. Infection <strong>of</strong> wounds
is an important complication that impairs wound healing.
A <strong>haemostatic</strong> <strong>agent</strong> should be bacteriostatic and/or
bactericidal, when used in surgical wound. In this study,
it was aimed to evaluate the potential antibacterial <strong>effect</strong>
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<strong>of</strong> ABS and FS under in vitro conditions.
Materials and methods
Ankaferd Blood Stopper®, ferric sulphate and 0.2%
chlorhexidine digluconate (control group) were screened
for antimicrobial activity by using well-diffusion
technique on agar plate.
Bacterial Strain and Culture Conditions
The antibacterial activity <strong>of</strong> each material was evaluated
against Staphylococcus aureus (S. aureus) ATCC#25923,
methicillin sensitive S.aureus (MSSA), methicillin resistant
S. aureus (MRSA), Enterococcus faecalis (E.faecalis)
(ATCC #29212), vancomycin resistant E. faecalis
(VRE), Candida albicans (C.albicans)(ATCC#10231),
Candida albicans oral isolate, Porphyromonas gingivalis
(P.gingivalis) (ATCC#33277), Lactobacillus acidophilus
(L.acidophilus) (ATCC#4356), Lactobacillus
salivarius (L.salivarius)(ATCC#11741), Streptococcus
mutans (S.mutans) (ATCC#25175), Streptoccous sobrinus
(S.sobrinus) (ATCC#33478) and Aggregatibacter
actinomycetemcomitans (A.actinomycetemcomitans)
(ATCC#29523).
S.mutans and S.sobrinus, were cultured in 5 ml <strong>of</strong> brain
heart infusion broth (BHIB, Oxoid, UK) at 37 ºC for
48 hours in an atmosphere <strong>of</strong> 5 % CO 2 . E.faecalis and
S.aureus were cultured in 5 ml <strong>of</strong> brain heart infusion
broth (BHIB, Oxoid, UK) at 37 ºC for 48 hours aerobically,
L.acidophilus and L.salivarius were cultured at
37 o C for 48 hours in 5 ml <strong>of</strong> MRS Broth (DeMan Rogosa
and Sharpe Medium, Merck, Germany) in an atmosphere
<strong>of</strong> 10% CO 2 . C.albicans was cultured in autoclaved-sterilized
Sabourraud Dextrose Broth (SDB, Oxoid,
UK) at 37 o C for 48 hours under aerobic conditions. A.
actinomycetemcomitans and P.gingivalis were cultured
in autoclave-sterilized trypticase soy broth (Oxoid, UK)
supplemented sheep blood (50 ml/L), Vit K (1µg/ml)
and hemin (5µg/ml) at 37 o C for 4-5 days in an automatic
anaerobic cabin (Electrotek AW400TG, UK) in the atmosphere
consisted <strong>of</strong> 90% N 2 , 5% CO 2 , 5% H 2 . The last
three ingredients were added to the main medium after
filter-sterilized with 0.22µm millipore. All <strong>of</strong> the freshly
grown bacterial suspensions in 5 ml <strong>of</strong> their specific broth
media as described before were adjusted to 10 8 cfu/
ml by using 0.5 Mc Farland test standard. For each <strong>of</strong>
the strains, 3 wells per each <strong>agent</strong> with a 5 mm diameter
were made under aseptic conditions in their specific agar
media containing petri plates for each <strong>of</strong> the strains separately.
Then the bacterial and fungal suspensions were
spread on to their specific agar plates.
The wells were filled with 20μl ABS (Ankaferd® Sağlik
Ürünleri A.Ş.) and Ferric sulhate (15.5%) (Astringedent®,
Ultradent Products Inc., Salt Lake City, UT,
USA). 0.2% chlorhexidine digluconate (CHX) was selected
as a positive control group, because <strong>of</strong> its widespread
clinical use; also it serves as a common point <strong>of</strong>

J Clin Exp Dent. 2012;4(3):e151-5. <strong>Antibacterial</strong> <strong>effect</strong> <strong>of</strong> <strong>haemostatic</strong> <strong>agent</strong>s.
reference for comparisons with other studies (13, 14).
Then, they were incubated on their specific media under
standard microbiological conditions according to the
growth properties as described before. After 48 hours the
inhibition zones around the wells were measured with
a digital caliper (Mitutoyo, SP, Brazil) for P.gingivalis
and A. actinomycetemcomitans and 24 hours for the rest
<strong>of</strong> the others, The measurements were repeated after 48
hours in the same manner. Tests were performed duplicate
for all <strong>of</strong> these <strong>agent</strong>s and materials. All inhibition
zones were measured at 24 and 48 hours by one author.
The results were statistically analyzed using One-way
analysis <strong>of</strong> variance and Tukey’s tests for the significant
interrelation between the groups using SPSS 13.0 for
Windows (SPSS Inc., Chicago, IL) with the level <strong>of</strong> statistical
significance set at p0.05). CHX
had the larger inhibition zone than ABS (p

J Clin Exp Dent. 2012;4(3):e151-5. <strong>Antibacterial</strong> <strong>effect</strong> <strong>of</strong> <strong>haemostatic</strong> <strong>agent</strong>s.
Discussion
This article has described the antibacterial activity in
vitro for <strong>new</strong>ly developed <strong>haemostatic</strong> <strong>agent</strong> ABS. In
this study, stains <strong>of</strong> Streptococcus mutans, Streptococcus
sobrinus, Lactobacillus acidophilus, Lactobacillus
salivarius, Staphylococcus aureus, Methicillin-resistant
staphylococcus aureus (MRSA), Methicillin-sensitive
Staphylococcus aureus (MSSA), Enterococcus faecalis,
Vancomycin-resistant Enterococcus faecalis (VRE),
Candida albicans, C.albicans oral isolate, Porphyromonas
gingivalis, Aggregatibacter actinomycetemcomitans
were used, which present in the oral cavity. S.mutans,
S.sobrinus, Lactobacilli species that are known as cariogenic
microorganisms play a major role in dental plaque
formation. S.aureus and E.faecalis are most commonly
seen hospital infection <strong>of</strong> surgical wounds and infections
associated with indwelling medical equipment. S.aureus
is also a human pathogen, and it causes systemic infections
such as wound infections, pneumonia, endocarditis,
and septicemia. Porphyromonas gingivalis and Aggregatibacter
actinomycetemcomitans play a major role
in periodontitis and periodontal tissue destruction.
Agar well-diffusion test is widely used in in-vitro method
<strong>of</strong> evaluation <strong>of</strong> antimicrobial activity <strong>of</strong> various
chemicals. The size <strong>of</strong> inhibition zone depends on the
solubility <strong>of</strong> the test material, time and temperature <strong>of</strong>
incubation (15). According to the other studies (6, 16),
agar well diffusion test was used in the present methodology
to verify the inhibition zone <strong>of</strong> the hemostatic
<strong>agent</strong>s, because it was clearly seen and reported that this
chemical did not dissolved in broth media such as Mueller
Hinton Broth.
Many <strong>haemostatic</strong> <strong>agent</strong>s and procedures have been
used for hemorrhage control such as ferric sulphate and
<strong>new</strong>ly developed ABS. ABS has been recommended in
the management <strong>of</strong> external and intraoral hemorrhage
such as pulpotomy, tooth extraction and flap operation in
dentistry. One <strong>of</strong> the most commonly used <strong>haemostatic</strong>
<strong>agent</strong>s in dentistry is ferric sulphate. The agglutination
<strong>of</strong> blood proteins result from the reaction <strong>of</strong> blood with
ferric and sulphate ions. This ferric ion-protein complex
mechanically seals the cut vessel and producing haemostasis.
By forming plugs that occlude the capillary
orifices, the protein complex also prevents the formation
<strong>of</strong> blood clots. Ferric sulphate has antibacterial <strong>effect</strong> on
all tested microorganisms in our study. This could be due
to acidic pH(17) and cytotoxicity (3) <strong>of</strong> ferric sulphate
solution. Lemon et al. (3) stated that although ferric
sulfate was an <strong>effect</strong>ive <strong>haemostatic</strong> <strong>agent</strong>, healing was
adversely affected. Jeansonne et al. (18) expressed that
ferric sulphate provides <strong>effect</strong>ive haemostasis, and also
irrigation with saline prior to closure <strong>of</strong> surgical defect,
does not delay healing.
Although the ABS has antibacterial activity, the inhibition
zones were found smaller than FS and CHX. Fe-
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rric sulphate showed inhibition zones against all <strong>of</strong> the
microorganisms, ABS, unfortunately, did not show any
inhibition against both <strong>of</strong> the lactobacilli species. There
was no statistical significance between ABS, ferric
sulphate and CHX for P.gingivalis (P>0.05). The zones
<strong>of</strong> bacterial growth <strong>of</strong> ferric sulphate and CHX became
smaller depending on time. On the other hand, the zones
<strong>of</strong> bacterial growth <strong>of</strong> ABS for some microorganisms
became larger depending on time. However, these differences
at 24- and 48- hour incubation for all tested
materials and control group were insignificant to make
inferences.
The <strong>effect</strong>iveness <strong>of</strong> ABS was due to the antimicrobial
<strong>effect</strong> <strong>of</strong> several contents. Fukai et al. (19) stated that
Glycyrrhiza glabra have antimicrobial activity against
S.aureus, MSSA and MRSA. Lee et al. (20) explain that
Alpinia <strong>of</strong>ficinarum has been used in Korea for several
hundred years to treat various infectious diseases. Agnihotri
and Vaidya(21) showed Thymus vulgaris has had
antibacterial <strong>effect</strong>. Similar to our findings, Tasdelen-
Fisgin et al.(6) reported that ABS has antibacterial activity
against methicillin resistant S.aureus (MRSA),
methicillin sensitive S.aureus (MSSA), and vancomycin
resistant E. faecalis (VRE).
Conclusions
In summary, a <strong>haemostatic</strong> <strong>agent</strong> should be bacteriostatic
and/or bactericidal, when used in surgical wound.
Based on the result <strong>of</strong> this study, FS had better antibacterial
activity than ABS under in vitro conditions. The
results <strong>of</strong> this study provide the information that FS and
ABS not only exhibit the <strong>haemostatic</strong> activity but also
antimicrobial activity. This may be an important finding
in terms <strong>of</strong> wound healing.
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